KR101018629B1 - PCR detection system for Prune dwarf virus - Google Patents

Abstract

The outbreak relates to a PCR test system for diagnosing plump atrophy virus (PDV) in cherry tree plants. The PCR test system includes an optimal primer combination for diagnosing PDV and a matching primer combination for the assay, and a species specific primer combination and a positive control that can support it.

Description

Plum atrophy virus PCAL detection system {PCR detection system for Prune dwarf virus}

The disease is contracted the virus Plum (Prune giving huge damage to crops in Cherry dwarf virus , hereinafter referred to as PDV). The PCR assay system includes a combination of optimal primers for diagnosing PDV and corresponding nested primers and species specific primer combinations and positive controls that can support them.

In the past, electron microscopy and serological methods were mainly used for virus diagnosis. Electron microscopy can confirm the presence of the virus, but its morphological features make it impossible to diagnose the species. Among serological methods, enzyme-linked immunosorbent assay (hereinafter referred to as 'ELISA') is about 1000 times lower in sensitivity than the most commonly used diagnostic method or PCR diagnostic method. Failure to diagnose due to poor nonspecific reactions often occurs.

PDV occurs naturally in rose and prunus (Prunes, peaches, cherries, etc.) plants in nature, and the means of transmission are contact infections by juice, insecticides such as beetle. PDV requires a diagnostic method that can detect it from Prunus spp., Which is imported as a quarantine pathogen. Until now, the enzyme-linked immunosorbent assay (hereinafter referred to as 'ELISA') has been mainly used to diagnose PDV. However, the diagnosis is often misdiagnosed by non-specific reactions of plant extracts and antiserum. In addition, low infection rates of PDV in the seeds to be tested are more likely to fail the ELISA test. Thus, in order to detect viruses in these seeds, it is generally necessary to diagnose PSI with 1000 times higher detection sensitivity than ELISA. The quarantine site requires the diagnosis of various pathogens on a single test sample, which requires a lot of labor and test costs. Therefore, the development of the same test method for each pathogen is necessary. When diagnosing by PSI, it is necessary to establish pathogen-specific diagnostic primer banks and to test for positive and negative reactions in case of difficulty in discriminating due to the low intensity of specific reactions or for non-specific reactions with other nucleic acids. You need a system that can. On the other hand, when isolate-specific primers are used for diagnosis, the test may fail, and therefore, development of a species-specific primer capable of detecting all isolates present in a viral species is required.

Currently, in the diagnosis of RNA virus, RT-PCR method with high detection sensitivity and convenience is most commonly used. Development of species-specific primers is the most important for PCR diagnosis of pathogens. No specific primers for diagnosing PDV have been patented.

 The present invention has been made to solve the above problems, and provides a PCR system of Plum Atrophy Virus (PDV), which has a high detection limit, can react with all PDV strains known to date, and can test both positive and negative responses. .

The present invention is a primer combination represented by SEQ ID NO: 11 and 23; Primer combinations represented by SEQ ID NOs: 11 and 25; Primer combinations represented by SEQ ID NOs: 11 and 26; Primer combinations represented by SEQ ID NOs: 12 and 22; Primer combinations represented by SEQ ID NOs: 12 and 23; A combination of primers represented by SEQ ID NOs: 16 and 23; And primer combinations represented by SEQ ID NOs: 19 and 32. Provides one or more primers for the diagnosis of prune atrophy virus selected from the group consisting of.

Preferably, there is provided a primer for diagnosing prune atrophy virus consisting of a primer combination represented by SEQ ID NO: 11 and 23 and a primer combination represented by SEQ ID NO: 12 and 22.

More preferably, the RT-PCR product of the primer combinations represented by SEQ ID NOs: 11 and 12 or the RT-PCR product of the primer combinations represented by SEQ ID NOs: 12 and 22 are assayed by the primer combinations represented by SEQ ID NOs: 16 and 29 It provides a primer for diagnosing plum atrophy virus.

According to another embodiment of the present invention, there is provided a prune atrophy virus diagnostic method using genomic RNA of a virus sample as a template and performing RT-PCR using the primer of claim 1.

Preferably, separating genomic RNA of the viral sample; Using the isolated genomic RNA as a template and performing RT-PCR using the primer of claim 1; And it provides a prune atrophy virus diagnostic method comprising the step of separating and analyzing the RT-PCR product by size.

In addition, preferably, after the step of separating and analyzing the PT-PCR product by size analysis using a primer for amplification of prune atrophy virus further comprising the step of performing PCR using the primers of the primers shown in SEQ ID NO: 16 and 29 to provide.

More preferably, it provides a method for diagnosing plum atrophy virus using the nucleotide sequence represented by SEQ ID NO: 33 as a positive control in the step of performing the PCR.

Provided are a plum atrophy virus diagnostic kit comprising the primer of claim 1.

Preferably, the plum atrophy virus diagnostic kit further comprises a positive control represented by SEQ ID NO: 33.

Within PDV species there are strains or isolates with slightly different base sequences. Therefore, if isolate-specific primers are used for diagnosis, there is a risk of diagnosis failure. In other words, the virus diagnostic primer should be species specific for each virus. Accordingly, the primers in the present invention were designed to work species specific. In the present invention, species-specific means that the primer can synthesize a specific RT-PCR product from the genome sequence of the entire virus to be diagnosed, and is common to all strains in the species of the virus to be diagnosed. And nonspecific reactions with nucleic acids from other similar species or plants should not occur.

In other words, the nucleotide sequences known to date have been collected within the species to search for common nucleotide sequences of all strains and isolates. The species-specific sequence was selected by excluding the nucleotide sequences of other viruses with a flexible relationship among the common sequences. From these species-specific sequences, sequences having optimal conditions as primers were designed as species-specific primers. The designed primers were subjected to the actual Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for each combination, and there was no non-specific reaction with nucleic acids other than the target virus. Selected.

In addition, the assay primer (nested primer) was also developed for the final selected primer combination.

In addition, by securing a variety of diagnostic primer combinations for PDV to build a primer combination that can be tested for PCR positive and negative reactions. The plasmid cloned the primer design region of PDV was prepared to obtain a positive control, and the nucleotide sequence was determined to confirm the positive reaction. In addition, PCR reaction conditions were identically developed in all virus and primer combinations to increase the test efficiency at quarantine sites.

Plum atrophy virus (PDV) fish test system can detect the PDV from plant seeds and tissues with a detection limit of 1,000 times more than conventional antiserum. PDV diagnostic primers included in the PSI test system have been developed to react with all PDV strains known to date, and also include tools for the validation of positive and negative responses. The test system of the present invention may be used to test PDV in industrialization and quarantine of plant virus RT-PCR diagnostic kits.

When the diagnostic method of the present invention is carried out, it is possible to precisely diagnose the presence of PDV from the seed or plant of Prunus vulgaris. It is also possible to industrialize RT-PCR diagnostic kits for plant viruses. By implementing the present invention it will be possible to minimize the damage of PDV in the country, it can be easily used at the border quarantine site can effectively block the influx of PDV-infected plants and seeds from foreign countries.

Hereinafter, the present invention will be described in more detail by way of examples.

Example 1 Design and Combination of PDV Diagnostic Primer

PDV consists of a single strand of RNA, and viral particles are spherical about 20 nm in diameter and oval about 20 to 38 nm in length. The genome of PDV consists of three parts. Taxonomically it belongs to the genus Bromoviridae Ilarvirus. PDV is mainly transmitted by juice, grafting and seeds. In nature, the host is associated with rose and prunus plants. The design of the diagnostic primers consisted of RNA 3 encoding the envelope proteins and the migration of the virus from which the subgenomic RNA was made.

 The search for the PDV common base sequence was made by multiple comparisons of the nucleotide sequences of 37 PDV isolates (Table 1) known to date. The selected common base sequences consisted of 14 ilavirus sequences that are taxonomically similar to PDV. A species specific sequence was searched in comparison with 2).

[Table 1] PDV isolate nucleotide sequence used for common base sequence analysis

[Table 2] Ilavirus sequencing used for species-specific sequencing

Of these species specific nucleotide sequences, 32 diagnostic primers satisfying primer conditions were designed (Table 3).

TABLE 3 Diagnostic primers designed from PDV species consensus sequence

Location * is Genbank ac. Created based on no.NC_008038.

The designed primer is shown in FIG. 1. The location is Genbank ac. no. Based on NC_008038. 35 primer combinations were made using these primers. RT-PCR products are shown in Table 4.

Table 4 PDV Diagnostic Primer Combinations and RT-PCR Expected Products

* Primer position is Genbank ac. Created based on no.NC_008038.

Example 2 Construction of Primer Combination for PDV Diagnosis and Selection of Optimal Primer Combination

PDV primers designed as shown in Table 4 were combined with 167, and the most effective primer combination was selected through RT-PCR assay with PDV and various related viruses. Selection of the optimal diagnostic primer combination consisted of three steps. The first is through RT-PCR with PDV infected plants, the second is nonspecific response analysis with bromoviride virus with taxonomic similarity, and the third is RT-PCR with virus associated with the host of PDV. Selection through analysis.

Total RNA isolation and RT-PCR methods used in the present invention are as follows.

(1) Total RNA Separation: Using a commercially available total RNA separation kit (method using phenol (Promega), TRIzol (Invitrogen Co.), easy-spin TM Total RNA Kit (iNtRON Co.)).

(2) RT-PCR: Reverse transcription reaction was 0.5μl total RNA isolated, 12.5pmole downstream primer, 5-fold reverse transcription buffer (250mM Tris-HCl, 150mM KCl, 40mM MgCl ₂ , 5mM DTT , pH 8.5) 5 μl of the reaction solution containing 1 μl, 10 μm dNTP 0.5 μl, 10 U RNase inhibitor, and 2 U AMV reverse transcriptase were incubated at 42 ° C. for 1 hour, and then denatured at 94 ° C. for 10 minutes. The polymerase chain reaction was carried out in 5 μl of reverse transcription reaction, 12.5 pmole upstream primer, 1.25 U Taq DNA polymerase, 10-fold polymerase chain reaction buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl _2). , pH 8.3) 2 mu l was added and distilled water was added to make the reaction solution 25 mu l. The reaction solution was amplified 40 times at 95 ° C 45 seconds, 55 ° C 60 seconds, 72 ° C 60 seconds and finally treated at 72 ° C for 7 minutes. RT-PCR products were identified by electrophoresis using 0.5 × TBE buffer and 1.5% agarose gel and stained with EtBr (ethidium bromide).

Figure 2 assays the specificity with PDV of the 167 primer combinations in Table 4. The number at the top of Figure 2 means the number of the combination. Total RNA extracted from PDV infected plum leaf tissue was used as RT-PCR template.

As shown in FIG. 2, among 167 primer combinations in the RT-PCR assay with PDV infected plants, 30 kinds having excellent reaction strength and no non-specific reactions that hinder diagnosis (combinations 6, 7, 10, 11, 27, 28, 31, 32, 46, 47, 48, 49, 50, 68, 69, 70, 83, 84, 85, 86, 98, 99, 100, 101, 112, 113, 114, 142, 163, 164) Primer combinations were selected.

The thirty primer combinations selected above were analyzed through a nonspecific reaction assay with three virus, Bromoviridae, which is a taxonomically similar virus (13 combinations 10, 27, 28, 32, 48, 49, 69). , 86, 100, 101, 142, 164) primer combination was selected. Figure 3 shows the RT-PCR analysis of secondary selected PDV diagnostic primer combination and host-associated virus. Nonspecific reaction assays include Alfalfa belonging to bromoviride mosaic virus (AMV), Tobacco streak virus (TSV), Cucumber mosaic virus (CMV) was used. In Figure 3, M is a size marker, lane 1 is PDV, lane 2 is AMV. Lane 3 is TSV and lane 4 is CMV.

The 13 combinations selected in the analysis of taxonomic pseudoviruses finally resulted in 7 primer combinations (combination 7, 27, 28, 32, 69, 86, 164) as a result of RT-PCR analysis with three viruses associated with the host. Selected.

Figure 4 shows the RT-PCR analysis of the third-selected PDV diagnostic primer combination and host-associated virus. As shown in FIG. 4, Combination 7 and Combination 27 of the 7 combinations showed no nonspecific response with the virus associated with PDV host. Three viruses were used to analyze nonspecific responses with host-associated viruses: Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Tomato ringspot virus (ToRSV). In FIG. 4, M is a size marker, lane 1 is PDV, lane 2 is ArMV, lane 3 is CLRV, and lane 4 is ToRSV.

On the basis of the above results, when diagnosing PDV from seeds or plants, RT-PCR is first performed using primer combination 7, 27 selected as an optimal diagnostic primer. If this diagnosis is insufficient, it may be verified using a combination of other primers of the PDV diagnostic primer combination.

Example 3 Example of Diagnosis and Verification of PDV

The primary diagnosis is using combination 7 and combination 27 as optimal primers for diagnosing PDV at the test site. If the assay of the first RT-PCR test result is required, i.e. to confirm that the RT-PCR product is a result from PDV, PCR is carried out using a nested primer for this primer combination. Here, the primer combination for assay for combination 7 and combination 27 is combination 134 (PDV-C80 / F60).

5 shows the results of RT-PCR analysis of the optimal primer combination for PDV diagnosis and primer combination for assay. In FIG. 5, M represents a size marker. Lane 1 shows the RT-PCR result of the optimal diagnostic primer combination 7 (739 bp), and lane 2 shows the RT-PCR result of the combination 134 (305 bp) as the assay primer combination for combination 7. In addition, lane 3 shows the RT-PCR result of the optimal diagnostic primer combination 27, and lane 4 shows the RT-PCR result of the combination 134 (305 bp) as the assay primer combination for the combination 27.

In this case, if the RT-PCR reaction result is derived from PDV, as shown in FIG. 5, the test PCR shows a positive reaction.

In addition, the nucleotide sequence of FIG. 7 (SEQ ID NO: 33) was prepared as a positive control including the entire region in which the primer was designed to easily identify the PCR product derived from the PDV primer combination of the present invention. This positive control was used as a positive control for PCR, and the base sequence of the inserted region of the positive control plasmid was determined and provided so that the base sequence of the required PCR product could be easily compared.

6 shows the PCR product of diagnostic primer combinations derived from PDV positive controls. In FIG. 6, M represents a size marker. Lane 1 shows PCR results of the PDV DNA fragment (PDV-C10 / FQ 50) inserted into the positive control (SEQ ID NO: 33; 1,065 bp), and lane 2 shows the PDV DNA fragment (PDV-C10 / FQ 60) inserted into the positive control. PCR result (945bp), lane 3 shows the PCR result (739bp) of the diagnostic primer combination 7, lane 4 shows the PCR result (673bp) of the diagnostic primer combination 27.

1 shows the location of primers for prune atrophy virus (PDV). Where the position of the primer is Genbank ac. no. It is based on NC_008038.

Figure 2 shows the RT-PCR results of 167 primer combinations for prune atrophy virus (PDV).

Figure 3 shows the RT-PCR results of the second selected primer combination and plum atrophy virus (PDV).

Figure 4 shows the RT-PCR results of the primers selected three and plump atrophy virus (PDV).

Figure 5 shows the RT-PCR results of primer atrophy virus (PDV) diagnostic primer combinations (combinations 7 and 27) and assay primer combinations.

Figure 6 shows the PCR product of a combination of diagnostic primers derived from plum atrophic virus (PDV) positive control.

Figure 7 shows the nucleotide sequence of prune atrophy virus (PDV) DNA fragment PDV-C10 / FQ50 inserted into the positive control.

<110> the Republic of Korea <120> PCR detection system for Prune dwarf virus <160> 33 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Prune dwarf virus <400> 1 ccaggtggaa gctcgaagty trt 23 <210> 2 <211> 21 <212> DNA <213> Prune dwarf virus <400> 2 gatgcttcac ggtccraatg t 21 <210> 3 <211> 21 <212> DNA <213> Prune dwarf virus <400> 3 gtccaaatgt rcccgtgaaa a 21 <210> 4 <211> 24 <212> DNA <213> Prune dwarf virus <400> 4 caaggacttt actgctgata ccaa 24 <210> 5 <211> 23 <212> DNA <213> Prune dwarf virus <400> 5 tgtcttccgt tactgccttg atg 23 <210> 6 <211> 24 <212> DNA <213> Prune dwarf virus <400> 6 ttccgttact gccttgatgt ttct 24 <210> 7 <211> 24 <212> DNA <213> Prune dwarf virus <400> 7 tgccttgatg tttctaatgg tgtc 24 <210> 8 <211> 24 <212> DNA <213> Prune dwarf virus <400> 8 ttgatgtttc taatggtgtc tacg 24 <210> 9 <211> 23 <212> DNA <213> Prune dwarf virus <400> 9 attaaaggtt tcgatgtgar tgc 23 <210> 10 <211> 22 <212> DNA <213> Prune dwarf virus <400> 10 gtggcaccta atcccctaca ac 22 <210> 11 <211> 24 <212> DNA <213> Prune dwarf virus <400> 11 gaagcttttg gtgtaacgat tggt 24 <210> 12 <211> 22 <212> DNA <213> Prune dwarf virus <400> 12 acgattggtt aactcacttt gt 22 <210> 13 <211> 21 <212> DNA <213> Prune dwarf virus <400> 13 aatagctcgt tttgtttacc a 21 <210> 14 <211> 24 <212> DNA <213> Prune dwarf virus <400> 14 ttcaccaatt tacttccaac tttc 24 <210> 15 <211> 24 <212> DNA <213> Prune dwarf virus <400> 15 cattaaatct ggaaagccta ctac 24 <210> 16 <211> 24 <212> DNA <213> Prune dwarf virus <400> 16 agctcggaag aataataata ctac 24 <210> 17 <211> 22 <212> DNA <213> Prune dwarf virus <400> 17 caggaccatt accggacaga cg 22 <210> 18 <211> 24 <212> DNA <213> Prune dwarf virus <400> 18 cagcgaggtg gatgatttct attc 24 <210> 19 <211> 21 <212> DNA <213> Prune dwarf virus <400> 19 ggaagatggc caagagcagt t 21 <210> 20 <211> 24 <212> DNA <213> Prune dwarf virus <400> 20 gactcattgc ataagaagaa acca 24 <210> 21 <211> 24 <212> DNA <213> Prune dwarf virus <400> 21 cgcgtttggt aattgggagt agtg 24 <210> 22 <211> 24 <212> DNA <213> Prune dwarf virus <400> 22 atccactgac tattytatcc atyg 24 <210> 23 <211> 20 <212> DNA <213> Prune dwarf virus <400> 23 tctttggcat cgagtgttgg 20 <210> 24 <211> 21 <212> DNA <213> Prune dwarf virus <400> 24 tggcatcgag tgttggaggt a 21 <210> 25 <211> 24 <212> DNA <213> Prune dwarf virus <400> 25 cgatcatacc agtaggagca agaa 24 <210> 26 <211> 22 <212> DNA <213> Prune dwarf virus <400> 26 ttgcacycca ctggcttgtt tc 22 <210> 27 <211> 20 <212> DNA <213> Prune dwarf virus <400> 27 actggcttgt ttcgctgtga 20 <210> 28 <211> 18 <212> DNA <213> Prune dwarf virus <400> 28 ccacaggcgc aytcacat 18 <210> 29 <211> 24 <212> DNA <213> Prune dwarf virus <400> 29 cgaaaccttt aatgagtccg taga 24 <210> 30 <211> 24 <212> DNA <213> Prune dwarf virus <400> 30 gtcaaggcag taacggaara caat 24 <210> 31 <211> 22 <212> DNA <213> Prune dwarf virus <400> 31 acatttggtc cgtgaagcat cc 22 <210> 32 <211> 24 <212> DNA <213> Prune dwarf virus <400> 32 raagctttgt gatcgggtag tagg 24 <210> 33 <211> 1065 <212> DNA <213> Prune dwarf virus <400> 33 gactcattgc ataagaagaa accatatgag aaaatgtacc caactttgag attcccgatt 60 gagaaatcgg aagcccttgc tgctgtggat gatgtaaaaa tactccaaac attcgccaaa 120 tcgcgattgg taattgggag tagtggaaag gttgatattg atcccagact tattgaaatt 180 aagtctgatg agtcaaagaa ggctttaaca gtagatttta agaatgttgc tgtacccgta 240 aagggtaaat cttcccttga cagtttcaag gaagccgagt ctgttcctct taaagaagag 300 aagtccgaca aggaagcttt tggtgtaacg attggttaac tcactttgtg agttaataat 360 tcgttttgtt taccattttc ttccaattct cgtttgttta tactctcata atgtctgaga 420 aagccattaa atctggaaag cctactaccc gatcacaaag ctttgcttta gctcggaaga 480 ataataatac taccccccct gctggacttg ttaagaaaca attcccgggt ggaagctcga 540 agtctgtttc cgagtggatg cttcacggac caaatgtgcc cgtaagaagt ttttccggta 600 tgatatctcg taccgagaat ctgacggtaa gttcgaccgc ttccggtgtt tattacacca 660 tgaaagtccg agagctcttc aaggactttt ctactgatac caaggtgtac ggaattgtat 720 tccgttactg ccttgatgtt tctaatggtg tctacggact cattaaaggt ttcaatgtga 780 gtgcgccagt ggcgcctaat cccctacaac gtaggaagtt cactgcgaaa caagccagtg 840 gggtgcaaat tctcgcccct actggtatga ccgtcgggga tataccagat gatctctggt 900 ttgtcataaa atatgacaat gcttttcagc ccgacgttcc ggtgtggttc tgtactcagt 960 acctccaaca ctcgatgccc aagagagttg agatccctga ttcagtgtta tacgctgaac 1020 gggatactgc tctcatggat gcgatggata gaatagtcag tggat 1065  

Claims (10)

Primer combinations represented by SEQ ID NOs: 11 and 23; Primer combinations represented by SEQ ID NOs: 11 and 25; Primer combinations represented by SEQ ID NOs: 11 and 26; Primer combinations represented by SEQ ID NOs: 12 and 22; Primer combinations represented by SEQ ID NOs: 12 and 23; Primer combinations represented by SEQ ID NOs: 16 and 23; And primer combinations represented by SEQ ID NOs: 19 and 32; at least one prune atrophy virus diagnostic primer selected from the group consisting of: a. The method of claim 1, Primer for prune atrophy virus consisting of a primer combination represented by SEQ ID NO: 11 and 23 and a primer combination represented by SEQ ID NO: 12 and 22. The method according to claim 1 or 2, Primer for prune atrophy virus, characterized in that the RT-PCR product of the primer combinations represented by SEQ ID NO: 11 and 23 are assayed by the primer combinations represented by SEQ ID NO: 16 and 29. The method according to claim 1 or 2, Primer for prune atrophy virus, characterized in that the RT-PCR product of the primer combination represented by SEQ ID NO: 12 and 22 are assayed by the primer combination represented by SEQ ID NO: 16 and 29. A genomic RNA of a virus sample as a template, and the prune atrophy virus diagnostic method characterized in that the RT-PCR is performed using the primer of claim 1. The method of claim 5, Isolating genomic RNA of the viral sample; Using the isolated genomic RNA as a template and performing RT-PCR using the primer of claim 1; And Plum atrophy virus diagnostic method comprising the step of separating and analyzing the RT-PCR product by size. The method of claim 6, After the step of separating and analyzing the PT-PCR product by size, the method of further comprising the step of performing PCR using the primers for the assay of the primer combinations shown in SEQ ID NO: 16 and 29. The method of claim 7, wherein Plum atrophy virus diagnostic method, characterized in that using the base sequence represented by SEQ ID NO: 33 as a positive control in the step of performing the PCR. Plum atrophy virus diagnostic kit comprising the primer of claim 1. 10. The method of claim 9, The prune atrophy virus diagnostic kit further comprises a positive control represented by SEQ ID NO: 33.

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